NO328812B1 - Agent for the Diagnosis of Tissue Proliferation Activity, Nucleoside Derivatives and Methods for Preparing a Radiolabeled Compound - Google Patents

Abstract

An agent which comprises, as an active ingredient, a radiolabeled compound as represented by the following formula or a pharmaceutically acceptable salt thereof: <CHEM> wherein R1 denotes hydrogen, or a linear- or branched-chain alkyl group having 1-8 carbon atoms, R2 denotes hydrogen, hydroxyl or a halogen substituent, R3 denotes hydrogen or fluorine substituent, R4 denotes oxygen, sulfur, or a methylene substituent, and R5 denotes a radioactive halogen substituent. The agent is stable in vivo, and either stays in cells or is incorporated in DNA, thus serving for diagnosis of tissue proliferation activity or treatment of proliferative disease. <IMAGE>

Description

Technical area

The present invention relates to a means for diagnosis of tissue proliferation activity, nucleoside denvat and method for producing a radiolabeled compound.

State of the art

If the proliferative activity of tumor cells can be determined non-invasively by means of imaging, this will help in assessing the growth rate and malignancy of tumours. Detection of the fastest growing regions of a tumor by imaging will be useful for planning radiation fields in radiotherapy, and identification of suitable parts for biopsy. Such methods will allow an early and accurate assessment of therapeutic effects, which are difficult to identify by CT- or MRJ-based anatomical assessment or PET-based measurement of glucose-metabolic changes. In particular, such methods will be useful for an early assessment of the therapeutic effects of anticancer agents that can cause strong side effects.

To solve these clinically important problems, the use of 5-iodo-deoxyundine labeled with radioactive iodine, and thymidine labeled with carbon-11 which is a positron emitter, has been investigated (Tjuvajev JG et al., J. Nucl. Med. 35 , pp. 1407-1417 (1994); Blasberg RG et al., Cancer Res. 60, pp. 624-635 (2000); Martiat Ph et al., J. Nucl. Med. 29, pp. 1633-1637

(1998); Eary JF et al., Cancer Res. 59, pp. 615-621 (1999); U.S. Patent No. 5,094,835; U.S. patent no. 5,308,605). It is believed that these radiolabeled compounds are taken into the cells as precursors for DNA synthesis necessary for cell division of rapidly growing tumors, and then phosphorylated by thymidine kinase, followed by incorporation into DNA, to reflect proliferative activity of the tumor. However, these radiolabeled compounds are rapidly cleaved in vivo, which makes it difficult to carry out non-invasive assessment of the proliferative activity of the tumor. The method that uses carbon-11-labelled thymidine requires particularly very complicated mathematical model analysis, and can hardly become popular as a diagnostic technique in nuclear medicine imaging.

The rapid metabolic cleavage of these radiolabelled compounds in vivo is thought to be due to cleavage of C-N glycosidic bonds by thymidine phosphorylase and instability of the labels in vivo. If the C-N glycosidic bonds are cleaved, the compound loses its affinity for tumours, thereby reducing the accumulation of radioactivity in tumours, while the radioactive metabolites increase the background radioactivity so that imaging of the tumors becomes difficult.

To solve these problems, radiolabeled compounds with metabolic stability have been synthesized by introducing fluorine atoms, which have high electronegativity, into the 2' or 3' position of certain nucleosides, and have been investigated for tumor imaging. Thus, 3'-deoxy-3'-fluorothymidine containing fluorine 18, a positron emitter, at the 3' position, shows a high stability m vivo and an accumulation in tumor tissue (Shields AF et al., Nature Med. 4, p. 1334 -1336 (1998)). Although this radiolabeled compound is stable z/z vivo, it is a radiolabeled compound with a short-lived positron emitter and therefore a cyclotron is required in the hospital, which limits the application of the compound. For this radiolabeled compound, the main process responsible for its accumulation in cells is the phosphorylation effected by thymid kinase which is an index of DNA synthesis and thus it does not serve as an agent that primarily reflects DNA synthesis.

A derivative of 5-iododeoxyuridine, in which fluorine is introduced at the 3'-position in the same manner as above to increase its stability in vivo, has recently been reported. However, although stable in vivo, this radiolabeled compound showed high retention in blood and failed to show a significant accumulation in a tumor compared to 5-iododeoxyuridine (Choi SR et al., J. Nucl Med. 41, p. 233 ( 2000).

2'-Fluoro-5-iodarabinouridine, in which fluorine is introduced at the 2'-position, shows a high stability in vivo, and has been used for the identification of introduction of, and expression in vivo of, a vector for gene therapy, using a phosphorylation reaction specific for thymidine kinase of human herpesvirus. The compound has also been used for imaging of viral infection, based on the high specificity of the viral thymidine kinase (Tjuvajev JG et al., Cancer Res. 56, pp. 4087-95 (1996); Tjuvajev JG et al, Cancer Res. 58, pp. 4333-4441 (1998); Wiebe LI et al., Nucleosides Nucleotides 18, 1065-1076 (1999); Gambhir SS et al., Nucl. Med Biol. 26, pp. 481-490 (1999); Haubner R et al., Eur. J. Nucl. Med. 27, p 283-291 82000); Tjuvajev JG et al, Cancer Res. 59, 5186-193 (1999), Bengel FM et al., Circulation 102, pp. 948-950 (2000)).

Against the background of the above-mentioned situation, the purpose of the present invention is to provide radiolabelled compounds which are practically useful in the clinical fields, stable w vivo, and have the ability to be back in the cells after being phosphorylated by thymidine kinase from mammals or reflect DNA synthesis activity after incorporation into DNA, especially those compounds labeled with a single photon emitter to achieve a wide spectrum of applications and the invention also aims to provide methods for the diagnosis of tissue proliferation activity and for the treatment of proliferative disease using agents containing the said radiolabelled compounds.

Description of the invention

In order to achieve the above objects, the inventors have synthesized a number of different radiolabeled compounds and have carefully investigated whether these are useful for imaging tissue proliferation activity. As a result, the inventors have found that radiolabeled inventions represented by the following formula can serve for diagnosis of tissue proliferative activity or treatment of proliferative disease, and have completed the present invention.

The present invention therefore provides a means for the diagnosis of tissue proliferation activity, characterized in that it comprises as active ingredient a radiolabeled compound represented by the following formula or a pharmaceutically acceptable salt thereof:

wherein R 1 denotes hydrogen or a linear or branched alkyl group of 1-8 carbon atoms, R 2 denotes hydrogen, hydroxyl or a halogen substituent, R 3 denotes hydrogen or a fluorine substituent, R 4 denotes oxygen, sulfur or a methylene substituent and R 5 denotes a radioactive halogen substituent, with the exception of the case where R 1 is hydrogen and R 4 is oxygen.

The radiolabeled compounds of the present invention are stable in vivo, and can remain in cells after being phosphorylated by mammalian thymidine kinase or reflect

DNA synthesis activity after they have been incorporated into DNA. They therefore enable effective diagnosis of tissue proliferation activity and treatment of proliferative disease, and are particularly useful as diagnostic radioactive imaging agents for diagnosis of tissue proliferation activity or as radioactive therapeutic agents for treatment of proliferative disease in accordance with internal radiotherapy, local radiotherapy or the like.

Accordingly, the agent can be used in a method for the diagnosis of tissue proliferation activity, comprising administering to a mammal an effective amount of a radiolabeled compound as represented by the above formula, or a pharmaceutically acceptable salt thereof, followed by in vivo imaging of the distribution thereof, and methods of treating proliferative disease, comprising administering to a mammal an effective amount of said radiolabeled compound or salt. Mammals include humans.

In the present invention, the radiolabelled compounds as represented by the above formula include salts thereof, or they may be in the form of a hydrate or solvate thereof. Such salts include pharmaceutically acceptable salts, for example a salt formed with a mineral acid, such as hydrochloric acid and sulfuric acid or with an organic acid, such as acetic acid. As such a hydrate or solvate can be mentioned the present radiolabelled compounds, salts thereof to which water molecules or solvent molecules have been attached. Furthermore, the compound of the present invention includes their various isomers as tautomers.

In the above formula, it includes linear or branched alkyl group with 1-8 carbon atoms as represented by Ri, for example methyl group, ethyl group, propyl group, t-butyl group and n-hexyl group, of which the methyl group is preferred. The halogen substituent represented by R 2 preferably includes fluorine, chlorine and bromine. R4 is preferably oxygen or sulphur, of which sulfur is particularly preferred.

The radioactive halogen substituent represented by R5 in the above formula is selected from the group consisting of F-18, Cl-36, Br-75, Br-76, Br-77, Br-82,1-123,1-124,1- 125,1-131 and At-211.

Preferred compounds as represented by the above formula include those wherein R| is hydrogen or methyl, R 2 is hydrogen or a halogen substituent, R 3 is hydrogen and R 4 is oxygen or sulphur, particularly preferred those in which R 1 , R 2 and R 3 are hydrogen, R 4 is sulphur, R 5 is a radioactive halogen substituent selected from F-18,1 -123,1-125 and 1-131.

Certain 4'-thionucleic acid derivatives represented by the above formula (wherein R5 is non-radioactive halogen substituent) have been reported to be resistant to bacterial thymidine phosphorylase as a result of studies of antiviral agents (Dyson MR et al., J. Med. Chem. 34 , pp. 2782-2786 (1991); Rahim SG et al., J. Med. Chem. 39, pp. 789-795 81996)). It has also been known that certain 5-iodo and 5-methyl-4'-sulfur substitution products inhibit phosphorylation of thymidine by human thymidine kinase (Strosselli S et al., Biochem J. 334, pp. 15-22 (1998)) . The chemical structures of these compounds with sulfur at the 4' position and their use as an antiviral agent are already known (International Publication WO9101326, International Publication WO9104982, Japanese Laid-Open Patent HEI 10-087687), but neither the corresponding radiolabeled compounds, or their use as a radiodiagnostic imaging agent or radiotherapeutic agent, have become known.

The chemical structures of certain compounds with a substituent at the 1'-position which

represented by the above formula (in which R5 is a non-radioactive substituent) and the production method thereof are previously known (Japanese Published Patent HEI 07-109289). However, neither the corresponding radiolabeled compounds nor their use as a radioactive diagnostic imaging agent, or a radioactive therapeutic agent, are known.

The compounds represented by a formula above can be used for various diagnoses of tissue proliferative activity and treatment for proferative diseases due to their in vivo stability and their ability to be retained in cells or to be incorporated into DNA.

Such diagnoses of tissue proliferative activity include, for example, diagnosis of hyperplasia, regeneration, transplantation or viral infection followed by abnormal proliferation.

The diagnosis of hyperplasia followed by abnormal proliferation includes, for example, the diagnosis of hyperplastic inflammation, benign tumors or malignant tumors. The diagnosis of the hyperplastic inflammation includes, for example, diagnoses regarding the activity of chronic rheumatoid arthritis and determination of therapeutic effects. The diagnosis of benign tumors includes, for example, diagnoses regarding location, activity and determination of therapeutic effects. The diagnosis of malignant tumors includes, for example, diagnoses regarding location, course, malignancy and determination of therapeutic effects, of primary and metastatic malignant tumors. Benign tumors include, for example, prostate hyperplasia, endometrial hyperplasia (cystic hyperplasia, adenomyosis uteri, hysteromyoma), ovarian tumor (cystadenoma), mammary gland (mastopathy, mammary gland fibroadenoma), pituitary adenoma, craniopharyngioma, thyroid adenoma, adrenocortical adenoma and pheochromocytoma. Malignant tumors include, for example, malignant lymphoma (Hodgkin's disease, non-Hodgkin's lymphoma), pharyngeal cancer, lung cancer, esophageal cancer, gastric cancer, colon cancer, hepatic cancer, pancreatic cancer, nephritic tumor (nephritic cancer, nephroblastoma), bladder tumor, prostatic cancer, testicular tumor, etc. -cancer, ovarian cancer, breast cancer, thyroid cancer neuroblastoma, heme tumor (primary brain tumor, metastatic brain tumor), rhabdomyosarcoma, bone tumor (osteosarcoma, metastatic bone tumor), Kaposi's sarcoma and malignant melanoma.

The diagnosis of regeneration accompanied by abnormal proliferation is exemplified by diagnosis of function of physiological regeneration of blood and diagnosis of pathological regeneration resulting from pathological loss of blood cells, such as assessment of physiological hematopoietic functions of bone marrow during treatment with anti-cancer drugs and diagnosis of pathological functions of the bone marrow in patients suffering from hypoplastic anaemia.

The diagnosis of transplantation accompanied by abnormal proliferation is exemplified by the diagnosis of blood cancer patients undergoing bone marrow transplantation, or very high dose chemotherapy using an anticancer agent, such as the diagnosis of successful introduction or proliferation of transplanted bone marrow cells in bone marrow transplantation.

The diagnosis of viral infection accompanied by abnormal proliferation includes, for example, the diagnosis of virus-infected lesions, and proliferation thereof in infectious diseases caused by type I or type II herpes simplex virus, varicella-zoster herpes virus, cytomegalovirus, Epstein-Barr virus or human immunodeficiency virus, in particular infectious diseases of the central nervous system (for example viral cerebritis, meningitis, etc.) caused by type I or type II herpes simplex virus or human immunodeficiency virus.

The treatment of prohferative diseases is exemplified by the treatment of malignant tumors or viral infection followed by abnormal proliferation. Such malignant tumors include, for example, malignant lymphoma (Hodgkin's disease, non-Hodgkin's lymphoma), pharyngeal cancer, lung cancer, liver cancer, bladder tumor, rectal cancer, prostatic cancer, uterine cancer, ovarian cancer, breast cancer, brain tumor (primary brain tumor, metastatic brain tumor) and malignant melanoma. Such viral infection includes infectious diseases of the central nervous system caused by type I or type II herpes simplex virus or human immunodeficiency virus, especially viral encephalitis or meningitis.

Methods for labeling the compounds represented by the preceding formula at the "5" position with a radioactive halogen can be carried out using known methods, such as methods using an isotope exchange reaction, and a method using a 5-chloromercury compound in which mercury is introduced into the "5" position of the compound or a 5-hydrogen compound in which there is no substitution at the "5" position of the compound. The method using the 5-chloromercury compound is already known as an iodine labeling method for the production of 5-iodo-2'-deoxyurtdine (US patent 4,851,520; Baranowska-Kortylewicz J. et al., Appl. Radiat. Isot. 39, p. 335

(1988)). However, this method is disadvantageous for the production of pharmaceuticals labeled with a radioactive nuclide with a short half-life due to side reactions (formation of "5-chloro" compounds, demercunization reaction), a long reaction time (6 hours) and formation of inorganic mercury compounds. The method using a 5-hydrogen compound is previously known as a method for the preparation of 5-iodo-2'-deoxyuridine from 2'-deoxyuridine (Knaus EE et al., Appl. Radiat. Isot. 37, p. 901 (1986) ; Fin RD et al., J. Labell. Comds. Radiopharm. 40, p. 103 (1997)). However, this method requires heating at 65-115°C, and is therefore not suitable for use with compounds that are easily decomposed under heating conditions, and cannot be said to be an ideal labeling method considering the properties of radioactive halogen atoms which preferably should not involve heating operations during the labeling reaction. Furthermore, the radiolabeling method using an isotope exchange reaction is also unsuitable for the production of pharmaceuticals that must be maintained at a certain quality level, due to the fact that the method is not capable of producing carrier-free labeled compounds and it is difficult to control variation of specific activity among different labeling series.

A further useful method of labeling the compounds represented by the preceding formula at the "5" position with a radioactive halogen is to allow a compound (5-trialkyltin compound) in which the pyrimidine base is replaced by a tnalkylstannyl group at the "5" position as represented by formula 11 in Figure 1, formula 21 in Figure 2, formula 28 in Figure 3, formula 40 in Figure 4, formula 50 in Figure 5 or formula 58 in Figure 6, to react with 0.1N sodium hydroxide solution of a radioactive halogen in a suitable solvent such as chloroform, so that the trialkylstannyl group at the "5" position is converted to a radioactive halogen substituent. This labeling method, which uses a 5-trialkyltin compound, is preferred because it does not suffer from such problems as with the above three labeling methods. Specifically, this method only requires a relatively short reaction time and does not produce "5-chloro" compounds or require heating as the reaction proceeds readily at room temperature. The resulting labeled compounds are free of carriers, and if a lower specific activity is desired, a labeled compound with a certain specific activity can be easily prepared by adding a carrier. This method is also valued in that purification after the reaction is easy to carry out. Specifically, 5-trialkyltin compounds are very different from the corresponding radioactive halogen-labeled compounds with respect to overall molecular polarity as the electrical properties at the "5" position differ between them due to the difference in the molecular polarity, labeled compounds and unreacted precursors can be easily separated using a commercial reverse phase silica gel cartridge after the labeling reaction. This allows for the elimination of the need for laborious purification by high pressure liquid chromatography.

According to a further aspect of the present invention, there is thus provided a method for the preparation of a radiolabeled compound represented by the following formula:

in which R 1 denotes hydrogen or a linear or branched alkyl group with 1-8 carbon atoms, R 2 denotes hydrogen, hydroxyl or a halogen substituent, R 3 denotes

hydrogen or fluorine substituent, R 4 denotes oxygen, sulfur or a methylene substituent and R 5 denotes a radioactive halogen substituent, except in the case that R 1 is hydrogen and R 4 is oxygen, characterized in that a nucleoside denate as represented by the following formula:

wherein R] denotes hydrogen or a linear or branched alkyl group of 1-8 carbon atoms, R 2 denotes hydrogen, hydroxyl or a halogen substituent, R 3 denotes hydrogen or a fluorine substituent, R 4 denotes oxygen, sulfur or a methylene substituent and R 5 denotes a trialkylstannyl group, except in the case that R| is hydrogen and R4 is oxygen, is reacted with an alkaline solution of a radioactive halogen in a solvent, whereby the trialkylstannyl group 1R5 is replaced with a radioactive halogen substituent.

5-trialkyltin compounds as represented by formula 11 in Figure 1, formula 21 1 Figure 2, formula 28 in Figure 3, formula 40 in Figure 4, formula 50 in Figure 5 and formula 58 1 Figure 6 are novel compounds that are useful intermediate compounds for the preparation of the radiolabeled compounds according to the present invention.

According to a further feature, the present invention provides a nucleoside derivative, characterized in that it is represented by the following formula:

wherein R 1 represents hydrogen or a linear or branched alkyl group of 1-8 carbon atoms, R 2 represents hydrogen, hydroxyl or a halogen substituent, R 3 represents hydrogen or a fluorine substituent, R 4 represents oxygen, sulfur or a methylene substituent and R 5 represents a tnalkylstannyl group, except in that case where R 1 is hydrogen and R 4 is oxygen.

In the above formula, they include linear or branched alkyl groups with 1-8 carbon atoms as represented by Ri, for example methyl group, ethyl group, propyl group, t-butyl group and n-hexyl group, of which the methyl group is preferred. The halogen substituent represented by R 2 preferably includes fluorine, chlorine and bromine. R4 is preferably oxygen or sulphur. The tnalkylstannyl group as represented by R5 including the trimethylstannyl group, the tnetylstannyl group and the tributylstannyl group.

Preferred compounds as represented by the above formula include those wherein R 1 is hydrogen or methyl, R 2 is hydrogen or a halogen substituent, R 3 is hydrogen, R 4 is oxygen or sulfur.

As can be seen from Figures 1-6, 5-trialkyltin compounds can generally be synthesized by

provide their corresponding halogen-containing compounds (as represented by formula 10 1 Figurej, formula 20 in Figure 2, formula 27 in Figure 3, formula 39 1 Figure 4, formula 49 1 Figure

5 or formula 57 1 Figure 6) as starting material, react the compound with bis(tnalkyl-tin) and bis(triphenylphosphine) palladium chloride in anhydrous 1,4-dioxane under heating

under reflux in an argon atmosphere, followed by purification.

Compound 10 (ITDU) in Figure 1 can be synthesized by a known method (formulas 1-8: Dyson, Mr et al., Carbo. Res 216, p. 237 (1991), and formulas 8-10: Oivanen, M et al., J Chem. Soc, Perkin Trans. 2, p. 2343 (1998)). Specifically, 2-deoxy-D-erythropentose (compound 1) is reacted with a 1% hydrochloric acid methanol solution to produce compound 2 which is then reacted with sodium hydroxide, tetrabutylammonium iodide and benzyl bromide to produce compound 3, in which hydroxyl groups are protected. The compound is reacted with α-toluenethiol and concentrated hydrochloric acid to produce compound 4 which is then reacted with triphenylphosphine, benzoic acid and diethyl azodicarboxylate to produce compound 5. Sodium methoxide is then used to remove the benzoyl group from compound 5 to produce compound 6, followed by its conversion to compound 7 using methanesulfonyl chloride. A ring is formed with sodium iodide and barium carbonate to produce compound 8 which is then reacted with 5-iodouracil in the presence of bistrimethylsilylacetamide and then with N-iodosuccinimide to produce compound 9. Then compound 9 is deprotected with titanium tetrachloride to produce compound 10.

Compound 20 (ITAU) in Figure 2 can be synthesized by a known method (Formula 13-17: Yoshimura Y et al., J. Org. Chem. 61, p. 822 (1996) and Formula 17-20: Yoshimura Y et al., J. Med. Chem. 40, p. 2177 (1997)). Specifically, 1,2;5,6-di-O-isopropylideneglycose (compound 13) is reacted with sodium hydride and benzyl bromide to produce a 3-benzyl compound which is then reacted with hydrochloric acid, aqueous sodium periodate solution and sodium borohydride to produce compound 14 , which is then converted with hydrogen chloride to compound 15. This compound is then reacted with mesyl chloride and sodium sulfide to produce compound 16 which is reacted with hydrochloric acid and sodium borohydride in sequence to produce compound 17. Hydroxyl groups are protected with sodium hydride and benzyl bromide (compound 18) and the resulting compound is converted to compound 19 with m-chloroperbenzoic acid (m-CPBA) and acetic anhydnd. The compound is further reacted with 5-iodouracil in the presence of 1,1,1,3,3,3-hexamethylenedisilazane (HMDS) to produce a glycosylated compound which is then reacted with boron chloride to produce compound 20.

The compound 27 in figure 3 can be prepared as follows. Compound 17 shown in Figure 2 is used as a starting material, which is reacted with t-butyldimethylsilyl chloride (TBDMSC1) in dimethylformamide (DMF) in the presence of imidazole to protect the hydroxyl group at the "5" position with a silyl group to produce compound 23. Trifluoromethanesulfonic anhydride ( Tf 2 O ) is added thereto in pyridine to produce compound 24 in which the hydroxyl group at the "2" position is trifluoromethanesulfonylated. The compound is reacted with potassium fluoride, together with "Kryptofix" (registered trademark) 222 and potassium carbonate in acetonitrile to produce a fluoride compound (compound 25) in which the substituent at the "2" position is stereochemically reversed. The compound is reacted with m-chloroperbenzoic acid (m-CPBA) in methylene chloride and further treated with acetic anhydride to produce compound 26. This is reacted with the product resulting from a reaction of 5-iodouracil and 1,1,1,3,3,3- hexamethylenedisilazane (HMDS) and with trifluoromethanesulfonic acid trimethylsilyl (TMSOTf). The resulting product is further treated with boron chloride in methylene chloride to afford compound 27.

The compound 39 (FIAU) in Figure 4 can be synthesized using a known method (formulae 30-37: Reichman U et al., Carbohydrate Res. 42, p. 233 (1975) and formulas 37-39' Asakura J et al. , J. Org. Chem. 55, p. 4928 (1990)). Specifically, compound 31 which has been synthesized in four steps from 1,2:5,6-di-O-isopropylidene glycose (compound 30) is treated with a cation exchange resin ("Amberhte" IR-120) to produce compound 32 which is then reacted with potassium penodate to produce compound 33. This is then reacted with sodium methoxide to produce compound 34, followed by acetylation of hydroxyl groups to produce compound 35. The compound is treated with a hydrobromide-acetic acid solution to produce compound 36, followed by condensation with a uracil derivative to produce compound 37. This is then reacted with diammonium cerium(III) sulfate (CAN) to produce compound 38, followed by deprotection of hydroxyl groups with sodium methoxide to produce compound 39.

Compound 49 (FITAU) in Figure 5 can be synthesized by a known method (formulas 42-46: Yoshimura Y et al., J. Org. Chem. 62, p. 3140 (1997) and formulas 46-49: Yoshimura Y et al., Bioorg. Med. Chem. 8, p. 1545 (2000)). Specifically, compound 43, which has been synthesized in nine steps from 1,2:5,6-di-O-isopropylideneglucose (compound 42), is reacted with diethylaminosulfur trifluoride (DAST) to produce compound 44, which is then reacted with m-chloroperbenzoic acid ( m-CPBA) to produce compound 45. This is then reacted with acetic anhydnd to produce compound 46 which is reacted with trifluoromethanesulfonic acid trimethylsilyl (TMSOTf) to effect condensation with 5-iodouracil derivative to produce compound 47. Finally the two protecting hydroxyl groups are removed to produce compound 49. Compound 57 (IMBAU) in Figure 6 can be synthesized by a known method (formulas 52-54: Itoh Y et al., J. Org. Chem. 60, p. 656 (1995) and formulas 55- 56: Asakura J et al., J. Org Chem. 55, p. 4928 (1990)), combined with known reactions for the protection and deprotection of hydroxyl groups (formulas 54-55 and formulas 56-57). Specifically, 1-[3-3,5-bis-0-(tert-butyldimethylsilyl)-2-deoxy-D-erythro-pento-1-enofuranosyl]uracil (compound 52) is reacted with pivalic acid and bromosuccinimide (NBS) to produce compound 53, which is then reacted with trimethylaluminum to produce compound 54. The protecting groups for hydroxyl groups are converted from tert-butyl-dimethylsilyl to acetyl, followed by reaction with diammonium cerium(III) sulfate (CAN) to produce compound 56. Finally, compound 56 is deprotected with ammonia to produce compound 57.

For radiolabeled compounds according to the present invention, appropriate dose and delivery methods should be chosen depending on the target diseases and purposes, but if they are used as a means of diagnosis of tissue proliferation activity, a radioactivity in the range of 37MBq to 740Mbq is added, preferably from 11 IMBq to 370MBq. Usually they are administered intravenously, but in some cases other routes of administration may be used, including arterial or intrapentoneal administration, and direct administration to a tumor or other affected parts.

If they are used as an agent for the treatment of proliferative disease, a radioactivity in the range of 37MBq to 7400MBq is added, preferably 185MBq to 3700MBq. They are usually administered intravenously, but in some cases other methods of administration can be used, including arterial or intraperitoneal administration and direct administration to a tumor or other affected parts. Furthermore, if used for therapeutic purposes, the above dose can be administered several times at appropriate intervals.

The means for diagnosis of tissue proliferation activity according to the present invention can serve for whole body or local scintigraphy and whole body or local SPECT imaging using nuclides for SPECT. When using nuclides for PET, they can also be administered for whole body or local PET imaging.

The means for diagnosis of tissue proliferation activity according to the present invention can serve for the quantitative determination of local proliferative activity based on appropriate model analysis. Furthermore, if non-proliferative tissue is used as a control, local proliferative activity can be easily defined in a semi-quantitative manner.

The agent for treating proliferative disease according to the present invention, when a beta emitter such as 1-131 is used therein, can serve to shrink large tumors by 1 cm or more in diameter, depending on the extent of radiation. When an alpha emitter such as At-211 is used, they can work on small lesions of 0.1 mm or less in diameter more effectively than a beta emitter, and are therefore expected to serve for the treatment of micro-metastases over the body. Furthermore, substances that emit Auger electrons, such as I-125, can have antitumor effects due to DNA breaks, but only after labeled compounds have gathered around the DNA. Therefore, suitable labeling nuclides for the treatment of systemic tumor sites including metastatic sites include alpha emitters such as At-211, and beta emitters such as 1-131 which may affect parts surrounding the sites depending on the extent. The most effective method is the cocktail therapy which uses a mixture of a compound labeled with an alpha emitter and a compound labeled with a beta emitter.

For treatment by local application, the compound is marked with nukhder that emmits Auger electrodes, such as 1-125, particularly effective for brain tumor that is difficult to remove completely by surgical operation and residual tumor from malignant melanoma, and on the basis of functional preservation breast cancer, rectal cancer, prostate cancer and malignant oral tumor, due to that they do no harm to parts other than pathologically proliferating cells due to the properties of rays emitted from them.

Methods of local delivery include, for example, a delivery into intracavitary sites such as colon cancer using an endoscope, a direct delivery to sites affected by a brain tumor during craniotomy and a delivery using a catheter into an artery relevant to the affected organ such as example liver affected by cancer.

Brief description of drawings

Figure 1 illustrates a synthesis process for a compound according to the present invention. Figure 2 illustrates a further synthesis process for a compound according to the present invention. Figure 3 illustrates a third synthesis process for a compound according to the present invention. Figure 4 illustrates a fourth synthesis process for a compound according to the present invention (5-trialkyltin compound) and [1-125] FIAU prepared therefrom. Figure 5 illustrates a fifth synthesis process for a compound according to the present invention. Figure 6 illustrates the second synthesis process for a compound according to the present invention. Figure 7 illustrates a diagram showing in vivo label stability of [I-125] ITDU and [T-125] ITAU measured in Example 16, along with [1-125] IUR as a control. Figure 8 illustrates a diagram showing in vivo label stability of [1-125] ITDU, [1-125] ITAU, [1-125] FITAU and [1-125] MB AU measured in Example 17, together with [1-125 ] IUR (high cleavability) as a control. Figure 9 illustrates a diagram showing in vivo distribution of [1-125] ITDU in normal mice measured in Example 18. Figure 10 illustrates a diagram showing in vivo accumulation of [1-125] ITDU, [1-125] ITAU, [1-125] FITAU and [1-125] IMBAU, together with [1-125] IUR as a control, in proliferating tissue measured in Example 18. Figure 11 illustrates a photograph (biological morphology) showing a scintigram of Walker tumor observed in example 19.

Examples

The present invention will be described below in detail with reference to examples.

Example 1

Synthesis of S-trimethylstannyl^-thio-l^deoxvuridine (compound 11)

As shown in Figure 1, benzyl-3,5-di-O-benzyl-2-deoxy-1,4-dithio-α,β-D-erythro-pentofuranoside (compound 8) was synthesized using 2-deoxy-D-erythro -pentose (compound 1) as starting material, according to the method of Dyson MR et al. (Carbo. Res. 216, p. 237 (1991)). Furthermore, 5-iodo-4'-thio-2'-deoxyuridine (ITDU: compound 10) was prepared from compound 8 according to the method of Oivanen M et al. (J. Chem. Soc, Perkin Trans. 2, p. 2343 (1998)). Compound 10 was then used as a starting material to produce 5-trimethylstannyl-4'-thio-2'-deoxyundine (compound 11) according to the following procedure.

Compound 10 (9.5 mg, 0.026 mmol), bis(trimethyltin) (17.3 mg, 0.052 mmol) and bis(triphenylphosphine)palladium(II) complex (5 mg) were dissolved in anhydrous 1,4-dioxane (3 mL) under an argon atmosphere and after heating under reflux for three hours, the mixture was concentrated under reduced pressure. The residue was purified by silica gel thin layer chromatography (chloroform-methanol, 6:1) to afford the target compound 11 (6.9 mg, 65%).

1H NMR (270Mhz, CD3OD) δ 0.26 (s, 9H, CH3Sn), 2.26 (ddd, 1H, J=4.6, 7.9, 13.2 Hz, 1H, H-2'), 2.27 (ddd, J=4.6, 6.6, 13 4 Hz, 1H, H-2'), 3.41 (m, 1H, H-4'), 3.71 (dd, J=5.9, 11.2 Hz, 1H, H-5'), 3.80 (dd, J=4.6 , 11.2 Hz, 1H, H-5'), 4.47 (q, J=4.0 Hz, 1H, H-3'), 6.41 (t, J=7.2 Hz, 1H, H-1'), 7.93 8s, 1H , H-5).

Example 2

Synthesis of fl- nsi- S- iod^- thio^- deoxvuridine (ri- 1251ITDU; compound 12)

To 0.1N sodium hydroxide solution was added (50 µl) of [I-125]-sodium iodide (33MBq), water (1 ml) and chloroform (1 ml), and then chloroform solution (4.7 µl) of iodine (60 µg, 0.47 umol) was added and the mixture was shaken for 10 seconds. After removing only the aqueous layer, ethyl acetate solution (100 µl) of compound 11 (100 µg, 0.25 µmol) was added and the resulting solution was allowed to stand at room temperature for 2 hours. A drop of 1N sodium thiosulphate solution was added and chloroform was evaporated. After addition of water (1 ml), the solution was passed through a "Sep-Pak Plus" QMA cartridge column. The column was washed with water (0.5 mL x 2) and the resulting aqueous solution was combined to afford I-125 labeled compound 12 (7.3 MBq, 22%).

Example 3

Synthesis of ri- mi- S- iod^- thio^- deoxvuridine ([ 1- 1231 ITDU: compound 12)

To 0.1% ammonium iodide solution (1 mL) containing [I-123]-ammonium iodide (2.0GBq), 1N hydrochloric acid (0.1 mL) and chloroform (1 mL) were added, and then a chloroform solution (4.7 µl) of iodine ( 60 µg, 0.47 u.mol) was added and the mixture was stirred for 10 seconds. After removing only the aqueous layer, ethyl acetate solution (100 µl) of compound 11 (100 µg, 0.25 µmol) was added and the mixture was allowed to stand at room temperature for 2 hours. A drop of 1N sodium thiosulfate solution was added and chloroform was evaporated. After addition of water (1 ml), the solution was passed through a "Sep-Pak Plus" QMA cartridge column. The column was washed with water (0.5 mL x 2) and the resulting aqueous solution was combined to afford I-125 labeled compound 12 (228MBq, 15%).

Example 4

Synthesis of 5-trimethylstannyl-1-(4-thio-D-arabinofuranosvuracil (compound 21) As shown in Figure 2, 1,4-anhydro-3-0-benzyl-4-thio-α-d-arabitol (compound 17) was synthesized from 1,2:5,6-di-O-isopropylideneglucose (compound 13) according to the method of Yoshimura Y et al (J. Org. Chem. 61, p 822 (1996)).Then, 5-iodo-1- (4-thio-D-arabinofuranosyl)uracil (ITAU: compound 20) prepared from compound 17 according to the method of Yoshimura Y et al (J. Med. Chem. 40, p. 2177 (1997)).This compound 20 was used as a starting material to produce 5-trimethylstannyl-1-(4-thio-D-arabinofuranosyl)uracil (compound 21) by the following procedure.

Compound 20 (4.0 mg, 0.010 mmol), bis(trimethyltin) (6.6 mg, 0.020 mmol) and bis(triphenylphosphine)palladium(II) chloride (5 mg) were dissolved in anhydrous 1,4-dioxane (5 mL) in a argon atmosphere and after heating under reflux for 4 hours, the mixture was concentrated under reduced pressure. The residue was purified by silica gel thin layer chromatography (25% methanol/chloroform) to afford the target compound 21 (2.3 mg, 55%).

1H NMR (270MHz, CD3OD) δ 0.7 (s, 9H), 3.55-3.67 (m, 1H), 3.77-3.95 (m, 2H), 4.07 (t, J= 5.9 Hz, 1H), 4.16 (t, J = 5.9, 1H), 6.28 (d, J=5.3 Hz, 1H), 8.03 (s, 1H).

Example 5

Synthesis of [ I- 1251- 5- iodo- l-( 4- thio- D- arabinofuranosvl) uracil ([ 1- 1251 ITAU:

(compound 22))

To a 0.1N sodium hydroxide solution (50 µl) of [I-125]-sodium iodide (67MBq), water (1 ml) and chloroform (1 ml) were added, and then a chloroform solution (4.7 µl) of iodine (60 µg, 0.47 umol) was added and the mixture was shaken for 10 seconds. After removing only the aqueous layer, ethyl acetate solution (100 µl) of compound 21 (100 µg, 0.24 mol) was added and the resulting solution was allowed to stand at room temperature for 2 hours. A drop of 1N sodium thiosulfate solution was added and chloroform was evaporated. After addition of water (1 ml), the solution was passed through a "Sep-Pak Plus" QMA cartridge column. The column was washed with water (0.5 mL x 2) and the resulting aqueous solution was combined to afford I-125 labeled compound 22 (17.3 MBq, 26%).

Example 6

Synthesis of 5-trimethylstannyl-1-(2-deoxy-2-fluoro-3-D- arabinopentofuranosyl)-uracil (compound 40)

As shown in Figure 4, blel-(3-3,5-di-0-acetyl-2-deoxy-2-fluoro-0-D-arabinopento-furanosyl)uracil (compound 37) synthesized from 1,2:5,6 -di-O-isopropylideneglucose (compound 30) according to the method of Reichman U et al., (Carbohydrate Res. 42, p. 233

(1975)). Furthermore, 5-iodo-1-(2-deoxy-2-fluoro-β-d-arabinopentofuranosyl)uracil (compound 39) was prepared from compound 37 according to the method of Asakura J et al. (J. Org. Chem. 55, p 4928 (1990)). This compound was used as starting material to prepare 5-trimethylstannyl-1-(2-deoxy-2-fluoro-B-D-arabinopentofuranosyl)uracil (compound 40) by the following procedure.

Compound 39 (5.0 mg, 0.013 mmol) bis(trimethyltin) (20.5 mg, 0.063 mmol) and bis-(triphenylphosphine)palladium(II) chloride (6.2 mg) were dissolved in anhydrous 1,4-dioxane (3 mL) under an argon atmosphere , and after heating under reflux for 2 h, the mixture was concentrated under reduced pressure. The residue was purified by silica gel thin layer chromatography (chloroform methanol 6:1) to afford the target compound 40 (3.6 mg, 66%). 1H-NMR (500MHz, CD3OD) δ 0.25 (S, 9H, CH3Sn), 3.72 (dd, J=5.0, 12.0Hz, IH, H-5'), 3.70-3.91 (m, H-4'), 4.33 (ddd, J=3.0, 5.0,18.5 Hz, 1H, H-3'), 5.02 (td, J=4.0, 53.0 Hz, 1H, H-2'), 6.25 (dd, J=4.5, 16.0 Hz, 1H, H-a'), 7.56 (S, 1H, H-5).

Example 7

Synthesis of 5-trimethylstannyl-1-(2-deoxy-2-fluoro-4-thio-B-D-arabinopento-furanosvDuracil (compound 50).

As shown in Figure 5, 1-0-acetyl-3-0-benzyl-5-0-(tert-butyldiphenylsilyl)-2-deoxy-2-fluoro-4-thio-D-arabinopentofuranose (compound 46) was synthesized from l ,2:5,6-di-O-isopropylidene glucose (compound 42) according to the method of Yoshimura Y et al. (J. Org. Chem. 62, p. 3140 (1997)). Furthermore, compound 46 was used to produce 5-iodo-1-(2-deoxy-2-fluoro-4-thio-B-D-arabinopentofuranosyl)uracil (compound 49) according to the method of Yoshimura Y et al. (Bioorg. Med. Chem. 8, p. 1545 (2000)). This compound was then used as a starting material to prepare 5-trimethylstannyl-2-(2-deoxy-2-fluoro-4-thio-(3-D-arabinopentofuranosyl)uracil (compound 50) by the following procedure.

Compound 49 (5.0 mg, 0.013 mmol), bis(trimethyltin) (16.9 mg, 0.052 mmol) and bis-(triphenylphosphine)palladium(II) chloride (6.0 mg) were dissolved in anhydrous 1,4-dioxane (3 mL) 1 an argon atmosphere and after heating under reflux for 1 3.5 hours was concentrated under reduced pressure. The residue was purified by silica gel thin layer chromatography (chloroform-methanol, 6:1) to afford the target compound 50 (1.9 mg, 35%).

IH-NMr (500MHz, CD3OD) 3 0.26 (S, 9H, CH3Sn), 3.61-3.68 (m, 1H, H-5'), 3.80-3.81 (m, H-4'), 4.37 (td, J= 6.0, 12.0 Hz, 1H, H-3'), 4.97 (td, J= 5.5,49.0 Hz, 1H, H-2'), 6 46 (dd, J=5.5, 11 5 Hz, 1H, H-l '), 7.99 (S, 1H, H-5).

Example 8

Synthesis of [ I- 1251- 5- iodo- 1-( 2- deoxyv- 2- fluoro- 4- thio- B- D- arabinopentofuranosvn-uracil ( H- 1251 FITAU:compound 51)

First, a 0.1N sodium hydroxide solution of [I-125]-sodium iodide (45 MBq) was concentrated, followed by addition of methanol (1 mL), addition of methanol solution (4.8 µL) of iodine (61 µg, 0.48 µmol) and shaking for 10 minutes. Then methanol solution (100 µl) of compound 50 (100 µg, 0.24 µmol) was added and the resulting solution was allowed to stand at room temperature for 2 hours. A drop of 1N sodium thiosulfate solution was added and methanol was evaporated. After addition of water (1 ml), the solution was passed through a "Sep-Pak Plus" QMA cartridge column. The column was washed with water (1.0 mL) and the resulting aqueous solution was combined to give 1-125-labeled compound 51 (3.5 MBq, 7.8%).

Example 9

Synthesis of 5-trimethylstannyl-l-methyl(2-deoxy-2-bromo-3-D-arabinopento-furanosvDuracil ([ 1-1251 IMBAU compound 58)

As shown in Figure 6, l-[2-bromo-3,5-bis-O-(tert-butyldimethylsilyl)-2-deoxy-1-C-methyl-B-D-arabinofuranosyl]uracil (compound 54) was prepared from l-[3 ,5-bis-O-(tert-butyl-dimethylsilyl)-2-deoxy-D-erythro-pento-1-enofuranosyl]uracil (compound 52) according to the method of Y et al. (J. Org. Chem. 60, p. 656 (1995)). Furthermore, 5-iodo-1-methyl(2-deoxy-2-bromo-3-D-arabinopentofuranosyl)uracil (compound 57) was prepared from compound 54 according to the method of Asakura J. et al., (J. Org. Chem. 55, p. 4928 (1990)). This compound was used as starting material to prepare 5-trimethyl-stannyl-1-methyl(2-deoxy-2-bromo-B-D-arabinopentofuranosyl)uracil (compound 58) by the following procedure.

Compound 57 (4.9 mg, 0.011 mmol), bis(trimethyltin) (16.0 mg, 0.049 mmol) and bis-(triphenylphosphine)palladium(II) chloride (5 mg) were dissolved in anhydrous 1,4-dioxane (3 mL) under an argon atmosphere, and after heating under reflux for 2.5 hours, the mixture was concentrated under reduced pressure. The residue was purified by silica gel thin layer chromatography (chloroform methanol, 6:1) to afford target compound 58 (3.8 mg, 72%).

1HNMR (500MHz, CD3OD) 8 0.25 (S, 9H, CH3Sn), 1.95 (S, 3H, 1'-CH3), 3.61-3.70 (m, 2H, H-5'), 4.08-4.11 (m, 1H, H-4'), 4.53 (d, J=3.0 Hz, 1H, H-5'), 4.79 (S, 1H, H-2'), 7.75 (S, 1H, H-5).

Example 10

Synthesis of | I- 1251- 5- iodo- 1- methyl-(2- deoxy- 2- bromo- B- D- arabinopentofuranosyl)-uracil ( H- 1251 IMBAUt compound 59)

First, 0.1N sodium hydroxide solution of [I-125]-sodium iodide (62 MBq) was concentrated, followed by addition of methanol (1 mL), addition of methanol solution (4.0 µL) of iodine (51 µg, 0.40 µmol) and shaking for 1 10 seconds . Then methanol solution (100 µl) of compound 58 (100 µl, 0.20 µmol) was added, and the resulting solution was allowed to stand at room temperature for 2 hours. A drop of 1N sodium thiosulfate slurry was added and methanol was evaporated. After addition of water (1 ml), the solvent was passed through a "Sep-Pak Plus" QMA cartridge column. The column was washed with vami (1.0 mL) and the resulting aqueous solution was combined to give 1-125-labeled compound 59 (8.3 MBq, 13%).

Example 11

Test for in vitro phosphorylation activity of fI-1251 ITDU and fI-1251 ITAU The phosphorylation activity of a labeled compound by thymidine kinase was determined using a crude enzyme extracted from the mouse lung cancer cell strain LL/2. Liquid enzyme was extracted from a LL/2 mouse lung cancer cell strain 1 the logarithmic

growth phase according to the method of Wolcott RM and Colacino JM (Anal. Biochem 178, pp. 38-40

(1989)). To the reaction liquid containing ATP, which is a phosphate donor, 2 nmol of the labeled compound and the liquid enzyme were added and reacted at 37°C for a specified period of time. The reaction was stopped by the addition of 1 ml of a 100 mm lanthanum chloride/5 mm triethanolamine solution. Phosphorylated material was prepared by centrifugal separation to form a phosphate-metal complex, followed by measurement of a radioactivity of the resulting precipitate using an automatic gamma counter (ARC-380, Aloka Co., Ltd.). Results are shown in Table 1 from which phosphorylation activity attributed to thymidine kinase was confirmed in both [1-125] ITDU and [1-125] ITAU.

Example 12

Test for in vitro metabolic stability of [ 1- 1251 ITDU and f 1- 1251 ITAU

To assess metabolic stability of the glycosidic bond, cleavage reactivity for thymidine phosphorylase from E. coli was investigated. To the reaction liquid, 2 nmol of the labeled compound and 9 units of a liquid enzyme (Sigma Corporation) were added, and the mixture was reacted at 25°C for a certain period of time, and the reaction was stopped by treatment in a boiling water bath for 3 minutes. The reaction liquid was subjected to centrifugal separation, and the supernatant was applied to a thin-layer silica gel plate together with an authentic standard (5-iodouracil:IU) and an unlabeled parental compound. This was developed with a mixture of chloroform and isopropyl alcohol (3:1), and then autoradiography was measured using a bioimaging analyzer (BAS-1500, Fuji Photo Film Co., Ltd.). The area of interest was set to peak components of the Rf value corresponding to the authentic standard, and the amount of resulting metabolite was calculated from its percentage abundance. The results are shown in Table 2 indicating that [1-125] ITDU and [1-125] ITAU are more stable than 5-iododeoxyundine ([1-125] IUR).

Example 13

Assessment of the in vitro stability of metabolism of different radioactive iodine-labelled nucleoside derivatives using thymidine phosphorylase In order to assess the metabolic stability of the glycosidic bond in different radioactive iodine-labelled nucleic acid derivatives, their cleavage reactivity for thymidine phosphorylase from E. coli was investigated. To the reaction liquid was added 0.5-12.0 nmol of the labeled compound and 0.0009-9.0 units of a liquid enzyme (Sigma Corporation), and the mixture was reacted at 25°C for a specified period of time, followed by treatment in a boiling water bath for 3 minutes to stop the reaction. As [1-125] IBMAU was unstable during heat treatment, the reaction liquid was cooled with ice to stop the reaction. The reaction liquid was subjected to centrifugal separation, and the supernatant was applied to a silica gel plate together with an authentic standard (5-iodouracil. IU) and an unlabeled parental compound. Development was done with a mixture of chloroform and isopropyl alcohol (3:1), and then the autoradiogram was measured using a bioimaging analyzer (BAS-1500, Fuji Photo Film Co., Ltd.). The area of interest was set to peak components of the Rf value corresponding to the authentic standard, and the amount of resulting metabolite was calculated from its percentage abundance. In the case of [1-125] FITAU and [1-125] IMBAU, a reversed-phase silica gel plate was used, and after development with a mixture of methanol and water (3:7), an autoradiogram was measured with a bioimaging analyzer (BAS- 1500, Fuji Photo Film Co., Ltd.) similar [1-125] IUR and others. Results of analyzes are shown in Table 3 which indicate that [1-125] FITAU and [1-125] IMBAU are even more stable than [1-125] JUR.

Example 14

Test of thymidine kinase-dependent incorporation into cells using thymidine kinase-deficient cells

Thymidine kinase-dependent incorporation of labeled compounds into cells was studied based on differences in incorporation between thymidine kinase-deficient cell lines L-M (TK-) and their parental L-M cells L-M and L-M (TK-) cells in the logarithmic growth phase were plated on 24-well plates each bearing 2.0 x 10<5> cells, and cultured overnight. Then 2 nmol of a radioiodine-labelled nucleoside derivative was added, and bh was incorporated into the cells for one hour. The cells were washed three times with an ice-cold phosphate buffer solution and dissolved in 0.1 NaOH, followed by determination of the degree of radioactivity incorporated into the cells using a bromine-type automatic gamma counter (ARC-380 or ARC-300, Aloka Co., Ltd. ). The measurements were analyzed to make judgments based on the amount of incorporated labeled molecules per unit weight of cellular proteins. Results are shown in Table 4 indicating that [1-125] ItdU and [I-125] FITAU were incorporated into cells in a thymidine kinase-dependent manner as in the case of [1-125] TUR as a control.

Example 15

Test for in vivo labeling stability of fI-1251 ITDU and 11-1251 ITAU

To assess the in vivo labeling stability of [1-125] ITDU and [1-125] ITAU, tests were conducted to study the accumulation of free iodine in the thyroid gland of normal mice. A 370KBq portion of each labeled compound was injected into each of 10-week-old normal mice in their tail vein, and three animals were sacrificed and dissected at appropriate intervals. For a control, in vivo distribution in [1-125] TUR was also observed. Incorporation of radioactivity into the thyroid gland was measured by a well-type automatic gamma counter (ARC-300, Aloka Co., Ltd.). Incorporated radioactivity in tissue was calculated as the delivered dose per gram of the tissue per unit time and represented in percentage, as shown in Figure 7. The results indicate that the accumulated radioactivity from [1-125] ITDU and [1-125] ITAU in the thyroid gland was significantly less than from the control in [1-125] TUR, which showed that the in vivo labeling stability of the agents is high.

Example 16

In vivo labeling stability of radioiodine-labeled nucleoside derivatives

To assess in vzvo stability of the deiodination against each radioactive iodine label

nucleoside denvat, tests were conducted to study the accumulation of free iodine in the thyroid gland of normal mice. 185KBq of each labeled compound was injected into each of 10-week-old normal mice (C57BL/6) into the tail vein, and three animals were sacrificed and anatomized with intervals longer than in example 15. Incorporation of the radioactivity i

the thyroid gland was measured with a well-type automatic gamma counter (ARC-300, Aloka Co., Ltd.). Incorporated radioactivity in tissue (%ID) was calculated as the amount supplied

per gram of the tissue and represented as a percentage, as shown in Figure 8). Results indicate that the accumulated radioactivity from [1-125] ITDU, [1-125] ITAU, [1-125] FITAU and [1-125] IMBAU in the thyroid gland was considerably less than that from the control in [1-125] IUR (highly metabolizable substance) which showed that the in vivo labeling stability of the agents is high.

Example 17

In vivo distribution of [ 1-1251 ITDU in normal mice

370KBq of [1-125] ITDU was injected into each of ten-week-old mice in the needle vein, and three animals were sacrificed and dissected at appropriate intervals. Incorporation of radioactivity into each tissue sample was measured with a well-type automatic gamma counter (ARC-300, Aloka Co., Ltd.). Incorporated radioactivity in tissue was calculated as the delivered dose per gram of the tissue, and represented as a percentage, as shown in Figure 9. Results indicate that the accumulated radioactivity in proliferating tissues, namely thymus and small intestine, was definitely higher than in non-proliferating tissues , namely brain tissue and muscle.

To assess the accumulation of each radiolabeled nucleoside derivative in proliferating tissues, tests were conducted to study in vivo distribution in normal mice. 185 Mbq of each labeled compound was injected into each of 10-week-old normal mice (C57BL/6) through their tail veins, and three animals were euthanized and anatomized at appropriate intervals. Incorporation of radioactivity into each tissue sample was measured using a well-type automatic gamma counter (ARC-300, Aloka Co., Ltd.). Incorporated radioactivity in tissue was calculated as the administered dose per unit weight of the tissue, and represented as a percentage (%ID/g). As shown in Figure 10, results indicate that [1-125] IUR (positive control) and [1-125] ITDU have accumulated in large amounts, especially in the thymus which is a proliferating tissue in normal young mice.

Example 18

Scintigraphy of Walker tumor using [ 1- 1231 ITDU

Malignant tumor, a typical proliferative disease, was lactate on scintigraphy. Walker tumor cells were transplanted subcutaneously into the right inguinal region of Wistar rats. After transplantation, 37 MBq of [1-123] ITDU was injected into the vena cava of rats suffering from an approximately 20 mm palpable tumor suitable for scintigraphy. Each tumor transplanted rat was anesthetized with "Ravonal" for four hours after the administration of a drug. Then, the rat was restrained in an upright position, and observed statically with gamma camera imaging equipment (GCA-90B, Toshiba Corporation). Imaging was performed using a high resolution medium energy collimator to obtain images over 10 minutes with a resolution of 256 x 256. Results are illustrated in Figure 11 showing that [1-123] ITDU serves for clear imaging of transplanted tumors ( indicated by an arrow) in Wistar rats.

Industrial applicability

The radiolabeled compounds of the present invention are stable in vivo, and they either return to cells after being phosphorylated by mammalian thymidine kinase or are incorporated into DNA to reflect DNA synthesis activity, and thus serve for diagnosis of tissue proliferation activity and treatment of prohferative diseases, in particular as radioactive diagnostic imaging agents for tissue proliferation activity diagnosis and as radioactive therapeutic agents for the treatment of proliferative disease by means of internal radiotherapy, local radiotherapy and the like.

Claims (1)

Means for the diagnosis of tissue proliferation activity, characterized in that it comprises as an active ingredient a radiolabelled compound represented by the following formula or a pharmaceutically acceptable salt thereof: wherein R 1 denotes hydrogen or a linear or branched alkyl group of 1-8 carbon atoms, R 2 denotes hydrogen, hydroxyl or a halogen substituent, R 3 denotes hydrogen or a fluorine substituent, R 4 denotes oxygen, sulfur or a methylene substituent and R 5 denotes a radioactive halogen substituent, with the exception of the case where R 1 is hydrogen and R 4 is oxygen. 2. Agent according to claim 1, characterized in that the radioactive halogen substituent in R5 is selected from the group consisting of F-18, Cl-36, Br-75, Br-76, Br-77, Br-82,1-123,1-124 ,1-125,1-131 and At-211. 3 Agent according to claim 1, characterized in that R4 is sulphur. 4. Agent according to claim 1, characterized in that Rj is hydrogen or a methyl group, R2 is hydrogen or a halogen substituent, R3 is hydrogen and R4 is oxygen or sulphur. 5. Agent according to claim 1, characterized in that R 1 , R 20 and R 3 are each hydrogen, R 4 is sulfur and the said radioactive halogen substituent R 5 is selected from the group consisting of F-18, 1-123, 1-125 and 1-131. 6. Means for diagnosis of tissue proliferation activity according to claim 1, characterized in that the diagnosis of tissue proliferation activity is a diagnosis of hyperplasia, regeneration, transplantation or viral infection followed by abnormal proliferation. 7. Means for diagnosis of tissue proliferation activity according to claim 6, characterized in that the diagnosis of hyperplasia followed by abnormal proliferation is a diagnosis of hyperplastic inflammation, benign tumor or malignant tumor. 8. Means for the diagnosis of tissue proliferation activity according to claim 7, characterized in that the diagnosis of hyperplastic inflammation is a diagnosis regarding the activity of chronic rheumatoid arthritis or the determination of therapeutic effects. 9. Means for diagnosing tissue proliferation activity according to claim 7, characterized in that the diagnosis of a benign tumor is a diagnosis relating to location, activity or determination of therapeutic effects. 10. Means for diagnosis of tissue proliferation activity according to claim 7, characterized in that the diagnosis of malignant tumor is a diagnosis regarding the location, course, malignancy or determination of therapeutic effects, of primary and metastatic malignant tumors. 11. Means for diagnosis of tissue proliferation activity according to claim 6, characterized in that the diagnosis of regeneration followed by abnormal proliferation is assessment of physiological hematopoietic functions of bone marrow during treatment with anticancer drugs, or diagnosis of pathological functions of bone marrow in patients suffering from hypoplastic anemia. 12. Means for diagnosis of tissue proliferation activity according to claim 6, characterized in that the diagnosis of transplantation followed by abnormal proliferation is diagnosis of acceptance or proliferation of transplanted bone marrow cells in bone marrow transplantation. 13. Means for diagnosis of tissue proliferation activity according to claim 6, characterized in that the diagnosis of viral infection followed by abnormal proliferation is diagnosis of virus-infected parts and proliferation thereof in infectious diseases caused by type I or type II herpes simplex virus, vancella-zoster herpes virus, cytomegalovirus, Epstein -Barr virus or human immunodeficiency virus. 14. Nucleoside denvate, characterized in that it is represented by the following formula: in which R] denotes hydrogen or a linear or branched alkyl group with 1-8 carbon atoms, R2 denotes hydrogen, hydroxyl or a halogen substituent, R3 denotes hydrogen or a fluorine substituent, R4 denotes oxygen, sulfur or a methylene substituent and R5 denotes a trialkylstannyl group, except in the case where R| is hydrogen and R4 is oxygen. 15. Nucleoside derivative according to claim 14, characterized in that R4 is sulphur. 16. Nucleoside derivative according to claim 14, characterized in that Ri is hydrogen or methyl, R? is hydrogen or a halogen substituent, R 3 is hydrogen and R 4 is oxygen or sulphur. 17. Nucleoside derivative according to claim 14, characterized in that the trialkylstannyl group 1 R5 is a trimethylstannyl group, a trimethylstannyl group or a tributylstannyl group. 18 Method for preparing a radiolabeled compound as represented by the following formula: in which Ri denotes hydrogen or a linear or branched alkyl group with 1-8 carbon atoms, R2 denotes hydrogen, hydroxyl or a halogen substituent, R3 denotes hydrogen or a fluorine substituent, R4 denotes oxygen, sulfur or a methylene substituent and R5 denotes a radioactive halogen substituent, except in the case that Ri is hydrogen and R4 is oxygen, characterized in that a nucleoside derivative as represented by the following formula: in which Ri denotes hydrogen or a linear or branched alkyl group with 1-8 carbon atoms, R2 denotes hydrogen, hydroxyl or a halogen substituent, R3 denotes hydrogen or a fluorine substituent, R 4 denotes oxygen, sulfur or a methylene substituent and R 5 denotes a tnalkylstannyl group, except in the case that R 1 is hydrogen and R 4 is oxygen, is reacted with an alkaline solution of a radioactive halogen in a solvent, whereby the tnalkylstannyl group in R 5 is replaced by a radioactive halogen substituent.